Process for the manufacture of physiologically active compounds

ABSTRACT

Producing 2-nitroimidazoles, such as azomycin, by oxidizing the corresponding 2-aminomidazole with Streptomyces eurocidicus var. nitroxydans.

United States Patent Inventors Giancarlo Lancini [51] ll. CL ....,I.:I..... (Dim/TF1 Pavia; [50] Field of Search 195/28, 80, Piero Sensi, Milan, both of Italy 51 App]. No. 638,584

Filed May 15,1967 References Cited Patented Nov. 16, 1971 1 UNITED STATES PATENTS Asslgnee fiP 1 3,317,401 5/1967 Nielson et a]. 195/51 Milan, Italy Priority May 9 Primary Examiner-Alvin E. Tanenholtz Great Britain Attorney-Joseph Hirschmann 22,168/66 PROCESS FOR THE MANUFACTURE OF PHYSIOLOGICALLY ACTIVE COMPOUNDS 6 Claims No Drawings ABSTRACT: Producing 2-nitroimidazoles, such as azomycin, US. Cl 195/28, by oxidizing the corresponding Z-aminomidazole with Streptol93/80, I9 5/5l myces eurocidicus var. nitroxydans.

PROCESS FOR THE MANUFACTURE OF PHYSIOLOGICALLY ACTIVE COMPOUNDS This invention relates to a new method for producing antibiotic substances. More particularly, the invention is concerned with the preparation of the known antibiotic azomycin and of its alkyl-substituted derivatives, of the general formula (Rn-k L NH wherein R represents a lower alkyl group and n is an integer from to 2 inclusive.

A primary purpose of this invention is to provide an advantageous process for preparing the known antibiotic substance, azomycin, and its homologues having antiprotozoal properties.

it is known that azomycin is produced by a micro-organism pertaining to the genus Nocardia, many characteristics of which resemble those of Nocardia mesenterica through the identity of the two micro-organisms could not be proved. The physiological and cultural characteristics of the azomycin producing strain are given in Journal of Antibiotics, 7A, 53 (1954).

Subsequently another azomycin producing micro-organism was found; it was named Streptomyces eurocidicus (Journal of Antibiotics, 8A, 105,1955).

We have now isolated from a sample of soil collected near lvrea (Italy) a Streptomyces which has now been filed with the American Type Culture Collection receiving the number l9,55l. The characteristics of said strain, hereinafter described, are quite similar to the ones of streptomyces eurocidicus. Moreover we have surprisingly discovered that the enzymatic activity of the new strain, which has been called Streptomyces eurocidicus var. nirroxydans ATCC l9,55l by us, can oxidize the amino group of some amino heterocyclic compounds to a nitro group. This effect allows the production of azomycin in high yield by cultivating Sfreptomyceseurocidicus var. nitroxydans ATCC 19,551 in a suitable culture medium and then introducing in said medium Z-aminoimidazole as starting compound, which can be easily prepared by known methods.

Moreover, we have discovered that not only azomycin can be prepared by this method, but that, if a substituted 2- aminoimidazole of the formula (RMwPJiNHZ wherein R and n have the above significance, is added to the medium wherein Streptomyces eurocidicus var. nitroxydans ATCC 19,551 is carrying is carrying out the oxidation from 2- aminoimidazole to 2-nitroimidazole, also the corresponding substituted 2-nitroimidazoles are produced in high-yield. This is a very valuable improvement, because it is known that azomycin and its substituted derivatives are not easily prepared by chemical processes. The process mainly consists in adding the starting compound to the culture medium when the fermentation has arrived at a suitable degree and continuing the fermentation at a temperature of -30 C. for l272 hours. A slight modification can be introduced by isolating and washing the mycelium at a suitable stage of its growth and suspend ing it in a fresh medium, to which the starting compound is then added. This method is preferably used when alkyl substituted imidazoles are to be prepared, because it decreases the concurrent formation of azomycin and makes the final purification easier.

The products are then recovered by conventional methods; the separation and purification are performed either chromatographically or, more advantageously, by a fractional crystallization.

The herein prepared alkylated ZnitroimidazoEs afieknown to possess, like azomycin, a high-activity against Trichomonas vaginalis. They are described for instance in ll Farmaco, Ed.Sci. 21,278 (1966) where also derivatives are reported having alkyl groups substituted on the nitrogen bearing a hydrogen atom. it is obvious that the herein prepared 2- nitroimidazoles can be useful also as intermediate compounds for obtaining the N-alkylated 2-nitroimidazoles, by entirely common procedures.

Streptomyces eurocidicus var. nitroxydansATCC l9,55l is herein below described.

General characteristics of Streptomyces eurocidicus var. nifroxydans ATCC 19,551 to microscopical examination.

Substrate mycelium fine, profusely branched, usually 0.8 to 1.0 uin diameter. Aerial mycelium usually well developed on the standard media, initially white becoming light gray. Sporophores long and wavy; spirals not formed.

In table I are reported the cultural characteristics of Streptomyces eurocidicus var. nifroxydans ATCC 19551. The culture media used were prepared according to Waksman (S. A. Waksman, The Actinomycetes, Vol. 2, The Williams and Wilkins Co., 1961). The results of the carbon utilization tests (Pridham and Gottlieb, J. BacterioL, 56, 107-114, 1948) are given in table 2 and, finally, in table 3 are given the results of some physiological tests.

TABLE 1.CULTURAL CHARACTERISTICS OF STREPTO- MYCES EUROCIDICUS VAR. NITROXYDANS ATCC 19651 Vegetative Soluble Media mycelium Aerlal mycelium pigment Oatmeal agar... Moderate growth, Moderate develop- Absent.

smooth surface; ment and hyaline. sporulation;

cottony surface; white. Bonnet agar Moderate growth, Moderate devel- Light brown. smooth surface; opment, poor light brown. sporulation,

cottony surface; ash gray to grey. Czapeck-Dox No growth a ar. Pot ato agar... Moderate growth, Moderate devel- Absent.

smooth surface; opment and hyaline. sporuiation,

cottony surface; white. Potato plug. Good lfiglowth. Traces of grayish Brown.

wri ed; white aerial hyaline. mycelium; no

spores. Glucose aspar- Moderate growth, Good development agine. smooth surface; and sporuiation;

h'yaline. cottony surface;

white.

D0 Moderate growth, Moderate devel- Pale yellow smooth surface, opment and to amber. yellowish. sporulation;

cottony surface; white. Nutrient agar. Moderate growth, Absent Brown.

smooth surface; brown to red brown with Starch Pmlet t d Absent agar... oorgrow o smooth surface; hyaline. Ca malate No growth Tyrosine agar- Poor growth, Absent Amber.

smooth surface; hyaline. Egg albumin ..do Moderate devel- Absent.

opment and sporulation; cottony surface; white. Cellulose agar. N 0 growth TABLE 2 Utilization of carbon compounds by strain Streptomyces eurocidicus var. nirroxydans ATCC 19,55 1.

Carbon sources Arabinose Xylose Glucose Galactose Fructose Mannose Rhamnose Lactose Maltese Sucrose Raffinose Gylcerol Sorbitol Mannitol Dulcitol lnositol Dextrin lnulin Starch Rihose Sorbose +H'good growth; ++moderate growth;:scarce growth; -no growth.

TABLE 3 Physiological characteristics of strain Streptomyces euridicus var. nitmxydans ATCC 1955 l.

Result Starch Hydrolysed Gelatine Slow a. partial hydrolysis Nitrates Not reduced Melanine Positive Litmus milk No changes The following nonlimitative examples illustrate the invention.

EXAMPLE 1 Preparation of Z-nitroimidazole A volume of 100 ml. of culture medium of the following composition (sterilized by heating at l20 C. for 20 minutes; pH after sterilization 6.5);

is inoculated with percent by volume of the fermentation broth apart prepared by cultivating the spores of Strepwmyces eurocidicus var. nitraxydans ATCC l9,55l in a suitable culture medium. The vessel is heated at 28 C. on a shaker (200 r.p.m.), and, after 48 hours,2-aminoimidazole is added in the amount of lOOY/ml. of the culture medium. Fermentation is carried out for 72 hours in the same conditions, then it stops; the broth is made slightly alkaline pH=8) with N sodium hydroxide and filtered on a suitable filter. The mycelium is repeatedly washed with water (pH=8) which is added to the filtrate, which is acidified with percent aqueous hydrochloric acid. The resulting solution is extracted three times with the same volume of ethyl acetate, and the obtained solution is concentrated in vacuo until a product crystallizes out; it is nomycin, which can be purified by recrystallization; the melting point is 283 C.

By adding lOOY/ml. of 2-aminoimidazole, an amount of 2- nitroimidazole corresponding to a concentration of 170Y/ml. in the final fennentation broth is recovered. A control culture wherein 2-aminoimidazole was not added, yielded a concentration of 68Y/ ml. of azomycin, so that the yield is 74 percent.

EXAMPLE 2 Preparation of 4(5)-methyl-2-nitroimidazole To 100 ml. of culture medium prepared and inoculated as described in example I, an amount of 4(5)-methyl-2- aminomidazole corresponding to lOOY/ml. is added after 48 hours of fermentation at 30 C. After 120 hours an amount of 1l8Y/ml. of nitroimidazoles (i.e.. comprising both 2- nitroimidazole and 4( 5 )-methyl-2-nitroimidazolel is extracted from the broth by ethyl acetate and separated with the method of fractional crystallization.

An amount corresponding to 55Y/ml. of 4(5)-methyl-2- nitroimidazoles and to 64Y/ml. of 2-nitroimidazole is ob- 5 tained. The m.p. of 4(5)-methyl-Z-nitroimidazole is 206 C.

EXAMPLE 3 Preparation of 4(5)-methyl-2-nitroimidazole A jar containing 40 l. of culture medium of the following is inoculated with 5 percent by vol. of the inoculum broth. The medium is kept at C. and mechanically stirred at 500 r.p.m. Aeration is provided by insufflating sterile air at a rate of 0.5 l. of medium and per minute. After 48 hours fermentation the mycelium is filtered, washed with a buffer solution of phosphates and suspended in 40 l. of the same solution (it is buffered at pH 6.5). An amount of 6.7 g. of 4(5)-methyl-laminoimidazole is then added and the fermentation is then carried out in the same conditions as above. After l2 hours the broth is extracted with ethyl acetate and the separation of the products is made by chromatography on a column of silicagel, using a 1:1 mixture of diethyl ether-petroleum ether as the eluting solvent. An amount of 4.5 g. of 4(5)-methyl-2 nitroimidazole (X(52 percent) is obtained; m.p. 206 C.

EXAMPLE 4 Preparation of 4.5-diethyl-2-nitroimidazole This compound is substantially prepared as above described inexample 2 for 4(5 )-methyl-2-nitroimidazole. The m.p. is 217 -l C.

EXAMPLE 5 to 7 l. A process for preparing a 2-nitroimidazole of the formula EJ NQ,

wherein R is a lower alkyl group and n is an integer from 0 to 2 .inclusive, which comprises subjecting a 2-aminoimidazole of the formula wherein R and n have the above significance; to the enzymic I var. nitroxydans is 2-aminoimidazole.

4. A process as in claim 1, wherein the compound subjected to the enzymic activity produced by Streptomyces eurocidicus var. nitraxydans is 4(5 )-methyl-2-aminoimidazole.

5. A process as in claim 1, wherein the compound subjected to the enzymic activity produced by Streptomyces eurocidicus var. nitroxydans is 4,5-dimethyl-Z-aminoimidazole.

6. A process as in claim 1, wherein the starting 2- aminoimidazole is contacted with the mycelium of Streptomyces eurocidicus var. nitroxydans, the product separated by filtration from the fermentation medium, and suspended in fresh medium. 

2. A process as in claim 1, wherein the starting compound is added to the fermentation medium of Streptomyces eurocidicus var. nitroxydans and fermentation is continued for 12-72 hours at 25*-30* C.
 3. A process as in claim 1, wherein the compound subjected to the enzymic activity produced by Streptomyces eurocidicus var. nitroxydans is 2-aminoimidazole.
 4. A process as in claim 1, wherein the compound subjected to the enzymic activity produced by Streptomyces eurocidicus var. nitroxydans is 4(5)-methyl-2-aminoimidazole.
 5. A process as in claim 1, wherein the compound subjected to the enzymic activity produced by Streptomyces eurocidicus var. nitroxydans is 4,5-dimethyl-2-aminoimidazole.
 6. A process as in claim 1, wherein the starting 2-aminoimidazole is contacted with the mycelium of Streptomyces eurocidicus var. nitroxydans, the product separated by filtration from the fermentation medium, and suspended in fresh medium. 